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REPRINT FROM 

SOIL SCIENCE 

RUTGERS COLLEGE 



ASSIMILATION OF ORGANIC NITROGEN BY ZEA MAYS AND 

THE INFLUENCE OF BACILLUS SUBTILIS ON 

SUCH ASSIMILATION^ 



A dissertation submitted in partial f ullillment of the requirements for the 
degree of Doctor of Philosophy in the University of Michigan 



By , 

Reed O. Brigham 



1 Publication No. 156, Botanical Department, University of Michigan. Reprint 
from Soil Science, vol. 3, no. 2, February, 1917. 






565 



D« Of BJ 
NOV ! 191f 



ASSIMILATION OF ORGANIC NITROGEN BY ZEAMAYS 

AND THE INFLUENCE OF BACILLUS SUBTILIS 

ON SUCH ASSIMILATION' 

By 

Reed O. Brigham, Instructor, University of Cincinnati 

Statement of Problem 

The aim of the work presented in this paper was first, to determine 
whether higher plants can utilize organic nitrogen directly without its be- 
ing acted upon by microorganisms ; second, to establish the relative im- 
portance of the compounds used ; and third, to determine how the utiliza- 
tion of organic compounds by plants is affected by the action of a bac- 
terium known to be able to decompose such compounds with the produc- 
tion of ammonia. The work embodies a series of experiments on the in- 
fluence of different nitrogenous compounds, in sterile and inoculated cul- 
tures, upon the growth of seedlings of tAVO varieties of Indian corn. 

The problem was carried out under the direction of Professor J. B. 
Pollock of the Botany Department of the University of Michigan, and the 
author wishes here to make grateful acknowledgement to him for his 
assistance. 

Historical Introduction 

The discussion of soil fertility in modern times has centered upon the 
nitrogen problem. Nitrogen has long been known as one of the elements 
necessary for plant growth and is the one which must most continually be 
provided to keep up soil fertility, because it exists in such small quantities 
in the soil and is so easily removed by crops or by natural processes. 

As long ago as 1835 Boussingault (5) showed that certain seeds con- 
tained as high as 4 to 7 per cent of nitrogen calculated on the dry weight 
basis. Later he (7) grew lupines, beans, and cresses in sand deprived of 
all nitrogen, and obtained about 1.3 per cent of nitrogen, showing prob- 
ably a minimum requirement of that element in the plants. In the growth 
of soil fungi under nitrogen starvation conditions, Goddard (13) obtained 
from 1 to 2 per cent of nitrogen in the mycelium. 

The growth of higher plants with an abundant supply of nitrogen 
shows that element to vary from 4.5 per cent in the leaves of red beets 
and in peas, 2.3 per cent in wheat grains, to 0.3 pe r cent in rye straw, ac- 

1 The data presented in this paper are from a thesis prepared in partial fulfillment of the re- 
quirements for the degree of Doctor of Philosophy in the University of Michigan. 
Received for publication October 25, 1916. 

(155) 



156 SOIL SCIENCE 

cording to Jost (17). With soil fungi grown in a rich nitrogenous 
medium, Goddard (13) found about 5 per cent in the mycelium. The 
analyses of different species of mushrooms, as given by Atkinson (1), 
shows the amount of nitrogen to vary from 2 to 6 per cent. 

The plant has three possible sources of nitrogen, namely, free nitrogen 
of the air and inorganic and organic compounds in the soil. The nitro- 
gen problem has centered around first one and then another of these 
sources, and in later times about the action of bacteria in relation to all 
three sources. 

The view was held from the time of Aristotle to about the end of the 
eighteenth century that humus was the source of all nourishment of 
plants, though the early Romans knew that the growing of leguminous 
crops on the fields in some way increased their fertility and they applied 
this knowledge to their argriculture. Following the discovery of the 
chemical elements the relation of these elements to the nutrition of plants 
became the subject of numerous investigations. 

The view of Aristotle dominated until about 1840 even though Ingen- 
houze thought plants were able to absorb free nitrogen from the air. At 
this time the great German chemist Liebig (26) concluded that plants 
absorb all or most of their nitrogen in the form of ammonium compounds, 
that the nitrogen problem was purely chemical, and that free nitrogen 
could not be utilized. He held firmly to these conclusions throughout his 
life. Liebig's opinion probably hindered further progress at this time, 
because he was recognized as one of the greatest chemists and his views 
were generally accepted. 

A new view was established about 1860, namely, that nitric acid or 
nitrates furnish an excellent, if not the most available source of nitrogen 
for the great majority of plants. This was given by Boussingault (6, 8, 
9) at the conclusion of experiments carried on from 1835 to 1860. 

The problem of the Leguminosae increasing the nitrogen was not ex- 
plained by these views of Liebig and Boussingault, and numerous experi- 
ments were carried out, among which were those of Lawes, Gilbert and 
Pugh (23). These early investigations finally culminated in the experi- 
ments of Hellriegel and Wilfarth (15). They showed in the^ltlearest way 
that microorganisms present in the soil are the cause of the formation of 
the nodules upon the roots of leguminous plants, and that when these 
nodules are present the assimilation of free nitrogen occurs. 

These conclusions in regard to bacterial action in the nitrogen prob- 
lem were followed in a short time by further proof of the role of bac- 
teria in nitrogen transformation. It was Winogradsky (56) in his bac- 
teriological studies, who ultimately cleared up the physiology of the nitro- 
bacteria, and his work has the right to be considered as one of the most 
important discoveries in plant physiology. He presented conclusive evi- 
dence of the existence of two kinds of nitrobacteria ; one of which pro- 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 157 

duced nitrites from ammonia, and the other nitrates from nitrites. This 
discovery gave a clearer understanding of the old views of Liebig and 
Boussingault, and showed how organic compounds can become the 
source of nitrogen after first being ammonified and nitrified. In this pro- 
cess organic nitrogen is changed to inorganic which then is available for 
direct assimilation either as ammonium compounds or nitrates. Wino- 
gradsky (57, 58, 59) also discovered the non-symbiotic nitrogen-fixing 
bacteria living in the soil and studied their characteristics. 

Organic Compounds 

The direct assimilation of organic nitrogenous compounds was a part 
of the old humus tlieory and was held until Liebig's chemical theory be- 
gan to prevail [Meyen (31) 1838], Experiments tending to prove direct 
assimilation of such compounds were first made in 1857 by Cameron (10), 
with positive results. About 10 years later Wolf and Knop (60) also did 
similar work, Baessler (2), Lawes and Gilbert (22), and Berthelot (3) 
also have done some valuable work along this line. Since in these early 
experiments the significance of bacteria was not understood and the 
necessit}^ for pure cultures was not recognized, all these early results are 
open to question. 

Recent work on the assimilation of organic nitrogenous compounds has 
taken into account the possible action of bacteria and various investiga- 
tions have indicated that these compounds are available for plants, al- 
though both negative and positive results have been obtained for the same 
compounds by various investigators. Strictly sterile conditions must be 
observed in testing accurately whether these compounds are directly as- 
similable or must first be acted upon by microorganisms to be ammonified 
or nitrified, or whether when so acted upon, they are rendered less toxic 
or more fully utilizable. There is also to be considered the difference in 
availability of the same substance for different plant species. 

Suzuki (52) found that yellow lupines, potatoes, wheat and Halesia 
hispidum produced more asparagin from urea than from ammonium salts, 
while barley did not ; and that, unlike nitrates, urea gave rise to asparagin 
in etiolated shoots. 

Pryanishnikov and Lyebyedyev (40) in 1897 carried out experiments 
in sterilized and non-sterilized media with hippuric acid, urea, leucin, as- 
paragin and aspartic acid. They found that none of the substances tested 
approached calcium nitrate as an effective source of nitrogen either in the 
sterilized or the non-sterilized media; also, that sterilization in all cases 
reduced the availability of the nitrogen of the organic substances, in most 
cases no gain being obtained in sterilized media. 

Nakamura (37) in making quantitative comparison of asparagin and 
ammonium succinate as sources of nitrogen for barley, onions and Asper- 



158 SOIL SCIENCE 

gillus oryzae, found that, in the case of the phanerogams, fully 50 per 
cent more growth was made where asparagin was added to the nutrient 
media than where the other compound was used. This wa% also true in 
the case of the fungus. 

In 1898 Lutz (30) carried out some very extensive experiments upon 
the assimilation of organic nitrogen. These experiments v/ere performed 
under sterile conditions, and thus fermentation products were excluded 
and nitrogen fixation prevented. The plants were grown in sterilized 
sand. The species used were, Cucurbita maxima, Zea mays, Cucumis 
prophetarum, Helianthus annuus, Ipomaea purpurea, Onicus benedictus, 
and Cucumis melo. 

Trimeihylamin, dimethylamin, monomethylamin, diethylamin, propy- 
lamin and butylamin were all assimilated by the plants without first being 
fermented in the soil, AUylamin and benzylamin were found to be un- 
favorable to the growth of phanerogamic plants. The phenol-animes were 
toxic and the hydramines and pyridin bases were not assimilated. Tetra- 
methylammonium and tetraethylammonium were not assimilated by phan- 
erogamic plants. Among the alkaloids he found that caffin and quinin 
were toxic and cocain, atropin and morphin were not available. 

Thompson (54) concludes from his studies with oats and barley that 
lu-ea and uric acid have the same value for the grasses as nitric nitrogen, 
urea being slightly better than uric acid. His results indicate, however, 
that hippuric acid is detrimental to plant growth. 

Pfeffer (39) has found that many heterotrophic organisms either re- 
quire a supply of peptone or other proteins or attain their maximum de- 
velopment only when thus supplied. Phanerogams and algae can also em- 
ploy as more or less valuable sources of nitrogen various organic sub- 
stcmces such as : urea, glycocoll, asparagin, leucin, tyrosin, guanin, uric 
acid, acetamid, but none is as favorable to grow^th as sodium nitrate. He 
has also found that hippuric acid is decomposed by plants into glycocoll 
and benzoic acid, the latter of which is useless. He believed that the parts 
of the plant where such decomposition occurs are probably the same as 
those in which proteins are synthesized. Pfeffer holds that under natural 
conditions phanerogams rarely absorb organic nitrogenous compounds. 

Schulze (49) quotes the investigations of a number of experimenters 
on the assimilation of leucin and tyrosin by plants and describes experi- 
ments of his own witli lupines, vetches and castor beans, which showed 
that these chemicals could be used as sources of nitrogen by phanerogams. 

Sawa (41), from investigations to determine if urea had any action 
on phanerogams, concluded that urea exercised an injurious action since 
the control plants made twice the growth of those in the solutions contain- 
ing urea, and the branches were more vigorous on the control plants. 

Kawakita (18) in his experiments on the efifect of guanidin on plants 
found that solutions containing 0.5 gm. of guanidin in 250 c.c. killed 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 159 

young barley plants in 3 days and that solutions one-fourth as concen- 
trated killed the plants in 2 weeks. 

MoUiard (33) studied the value of asparagin and urea because, as he 
says, the assimilation of these two substances has been reported by others 
with different results. He grew his plant under sterile conditions and 
concludes from his experiments that these two substances maintain a nu- 
trient role for higher plants. 

Lefevre (24) in a series of experiments conducted with plants grown 
without carbon dioxide, found that glycocoll, alanin, tyrosin, and leucin 
not only furnish nitrogen, but also furnish the carbon required, 

Schreiner and Reed (44) in their extensive studies tried guanin, al- 
though it is only slightly soluble in water. They vised it in amounts vary- 
ing from 1 to 40 parts per million, and in all of these concentrations it bad 
a slightly beneficial effect upon the growth of wheat plants. 

Guanidin carbonate, however, when tested on wheat plants in distilled 
water showed a very strong toxicity. When this solution was treated with 
carbon black, not only was the toxic action counteracted but the plants 
gave a better growth than the check in distilled water. 

In a later publication (45) the same authors in their experiments fotmd 
guanidin carbonate even in solutions so dilute as one part per million suf- 
ficient to kill wheat seedlings. Guanin was not harmful. Their experi- 
ments showed further that for wheat seedlings leucin and asparagin are 
not at all toxic. Alanin and glycocoll were slightly injurious at higher 
concentrations. Cumarin was extremely poisonous. 

Bierema (4) reported that formamid and acetamid were not readily 
assimilated, although the latter was capable of supplying both nitrogen 
and carbon. Guanidin carbonate alone was not actively assimilated, but 
was somewhat more readily taken up in the pressence of calcium lactate, 
sucrose and glycerol. Uric acid was completely converted into ammonium 
carbonate, but less readily into urea. Leucin and tyrosin, especially the 
first, were readily assimilated, ammonium acetate more readily, especially 
in the presence of dextrose, and ammonium butyrate was still more 
readily assimilated. 

Molliard (34) in further researches upon the utilization of organic 
nitrogen by higher plants, grouped his investigations under three main 
heads: (a) the action of various organic nitrogenous substances on the 
development and production of green and dry matter; (b) the total nitro- 
genous content of plants thus grown, and (c) the formation of protein 
substances from the absorbed nitrogen. 

The following substances were used in the culture media in the ratio 
of 1:1000 parts: urate of sodium, aspartic acid, asparagin (1:500), gly- 
cocoll, legumin, cyanide of sodium, amygalin, hydrocyanic acid, leucin, 
tyrosin, myronate of potassium and alanin. Of these substances the first 
nine were utilized by the plants as shown by the increase in green and 



160 SOIL SCIENCE 

dry matter over similar plants grown as checks. This utilization was the 
greatest in the case of urate of sodium, and decreased in order named 
down to leucin. Tyronsin, myronate of potassium and alanin were toxic to 
the roots only. The amount of protein nitrogen foimd in seedlings grown 
in the presence of asparagin and glycocoll was about twice the total nitro- 
gen of the ungerminated seeds. 

Hutchinson and Miller (16) in their work conclude that, while peptone 
and certain other nitrogenous compounds may be taken up and to some 
extent utilized by plants, they are unable to furnish the whole of the nitro- 
gen required, or at any rate, to supply it with sufficient rapidity. They 
further conclude that their results are not sufficiently numerous to make 
it possible to trace any connection between the assimilability or non-assim- 
ilability of nitrogenous compounds and their constitution. They found it 
impossible to adhere to their original intention of sterilizing the media, for, 
although sterile media were most suitable, their employment was pre- 
vented by the impossibility of sterilizing many of the most desirable sub- 
stances without more or less decomposition. They grouped the com- 
pounds experimented with under five heads, namely: (a) readily assimi- 
lated — ammonium salts, acetamid, urea, barbituric acid (with calcium 
carbonate), alloxan, humates; (b) assimilated — formamid, glycin, a. 
aminopropionic acid, guanidin hydrochloride, cyanuric acid, oxaraid, 
sodium asparatate, peptone; (c) doubtful — trimethylamin (contrary to 
the results of Lutz), papa-urazine, hexamethylenetetramin ; (d) not as- 
similated — ethyl nitrate, propionitrile, hydroxy lamin hydrochloride, 
methyl carbamate; (e) toxic — tetranitromethane. This grouping, they 
affirm, is applicable only when peas are used and, as the authors suggest, 
it is possible that other plants may be able to utilize some of the sub- 
stances which with peas have given negative results. Glycocoll in one 
culture gave an increase and in another a decrease. 

Kossowicz (21) in his studies upon the assimilation of guanin and 
guanidin by mould fungi, found of about 10 fungi experimented with 
that all were able to utilize guanin as a nitrogen source, also guanidin un- 
der the conditions favoring the formation of ammonia. It is of interest 
that these results are different from those with the higher plants. 

Schreiner (43) in his researches found that when creatin and ni- 
trates are present less nitrates are used by the plant, although a larger 
plant growth takes place. The plant absorbs the creatin and builds it 
into its tissues. The author states that, upon his rather extensive inves- 
tigations, he is ready to formulate the theory that the degeneration pro- 
ducts of protein are absorbed directly by the plant from the soil and that 
the plant uses these units for building up the complex proteins as far as 
it is possible to do so. Since the plant must spend much energy in the 
building up of nitrates into amido groups of protein molecules, it is rea- 
sonable to suppose that the unit part of the complex molecule, when pre- 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 161 

sented to the plant, will be used by it in preference to expending labor on 
the nitrate. The use of these decomposition products gives a different 
point of view to the problems of soil fertility. 

Skinner (50) has shown in his experiments that the action of crea- 
tinin and creatin on growth is very similar. They had a beneficial effect 
on the growth where nitrate nitrogen was lacking and where only small 
amounts of nitrate were present, but when large amounts of nitrates were 
present these compounds produced no effect. 

Skinner and Beattie (51) report that in all the plants experimented 
with asparagin is beneficial to growth, even when nitrate is present, al- 
though to a lesser degree. 

Schreiner and Skinner (46) report upon some of the nitrogenous soil 
constituents as follows : Guanin at a concentration of 40 parts per mil- 
lion showed an increase in growth of 5 per cent over that of the growth 
in a distilled water control, and good root development. Asparagin 
showed, both in cultures with and without other sources of nitrogen, a 
decidedly beneficial effect upon the growth of plants. Guanidin produced 
a very decided toxic influence on growth. Glycocoll ( amido-acetic acid) 
in water solutions was found to be beneficial. Alanin, in lower concen- 
trations, was beneficial to growth, although in concentrations as high as 
500 parts per million it slightly injured the roots of wheat seedlings, 

Dachnowski and Gormley (12) in studies on bog plants and transpira- 
tion, togetlier with the effect of glycocoll, state that tlie glycocoll is in 
part undoubtedly the gycocoll absorbed and assimilated. 

Schreiner and Skinner (47) in experimenting upon the action of 
methyl glycocoll and glycocoll, found that the first was harmful, and the 
latter beneficial to the growth of plants. 

It will be seen from the above review of the work on tliis subject that 
there is a great deal of contradiction in results obtained by different 
workers. 

Bacterial Action 

The process of nitrification was first shown in 1877 to be dependent 
upon the presence of certain micoorganisms, by Schloesing and Miintz 
(42). In 1893 Miintz and Coudon (36) showed for the first time that 
ammonia production in the soil is due to bacteria. However, in 1862 
Pasteur (38) was the first to prove that the formation of ammonia from 
urea was brought about by the action of mocroorganisms. Within the 
last twenty years the work of numerous investigators shows that am- 
monia production from organic nitrogen is a function of most of the soil 
bacteria. Among the soil bacteria with this capacity is Bacillus subtilis. 

Miquel (32) shows in his numerous experiments the effect of some 
of the species of bacilli which play an important role in the ammonifying 
of urea and splitting of uric acid into urea and other compounds. In 
(iii— 13^ 



162 SOIL SCIENCE 

his conclusion he suggests that this sphtting may play some part in the 
availability of these substances for the growth of plants. 

Lohnis (29) found that soil bacteria rapidly convert urea into ammon- 
ium carbonate, probably by the action of Urobacillus Pasteurii. 

The experiments regarding the decomposition of uric acid by bacteria, 
carried on by Liebert (25) showed that by aerobic bacteria the acid was 
broken up into carbon dioxide, ammonia, and the intermediate products^, 
allantoin, urea, and oxalic acid. 

Lipman (27) has recently determined that B. subtilis changes about 
19 per cent of nitrogen present into ammonia. 

Kelly (19, 20) has recently made extensive studies upon the bio- 
chemical decomposition of nitrogenous substances and ammonification, 
using commercial products such as casein, dried blood, cottonseed meal 
and linseed meal. The results showed that the different materials were 
converted into ammonia at greatly different rates and amounts. 

New Experiments 
In view of the contradictory results found by different investigators 
on the assimilation of organic nitrogen and in view of the desirability of 
testing more species of plants for their capacity- of assimilating organic 
compounds, it was considered worth while to undertake the study of this 
problem with Mays plants grown with their roots in media free from 
bacteria except such as were intentionally inoculated into the cultures. 
The bacterium chosen was B. subtilis. This choice was so made because 
it is one of the common and widely distributed soil bacteria and has been 
shown to be capable of ammonifying organic compounds. 

Methods and Techniqe 

It is of the greatest importance that sterile, bacteria-free cultures be 
employed in investigations of soil bacteria, and especially in the case of 
experiments relative to the availability of organic nitrogen, for only thus 
can nitrification and ammonification be certainly prevented. 

It is first necessary to select a medium which may be kept absolutely 
sterile throughout the experiment and which will permit the plants to 
make an active growth during a long period. Three media suggest them- 
selves, namely, sand, water, and agar. Cultures in which each of these 
was employed were experimented with, and the latter was found to be the 
best adapted to the work at hand. 

The plants which were placed in the sand cultures made a very poor 
growth and seemed to show evidence of a toxic influence. Warington 
(55) claims that such a material is in several respects a very unnatural 
medium for plant growth and is generally unsuited for this kind of inves- 
tigation. Furthermore, it has so low a Avater-holding capacity that a cul- 
ture when saturated contains about 60 per cent of inert material. Be- 
cause of the small amount of water, some means of supplying sterile water 



BRIGHAM—ASSIMILATION OF ORGANIC NITROGEN 163 

during the growth must be devised and this adds to the danger of con- 
tamininating the culture with fungi and bacteria. 

Water cultures have been found satisfactory for a great deal of work 
by physiologists, but they require frequent change for the best results, 
and this is impractical under sterile conditions. Some means of aeration 
may be used. However, this can be done only at intervals, for contin- 
uous aeration is not practical with large numbers of cultures. Combes 
(11) suggests a method of aeration at intervals, but it requires special 
culture jars not easily obtainable. A preliminary experiment showed the 
poor growth of Zea plants in non-aerated water cultures. 

Of the three media mentioned, the agar seemed then to afford the best 
substratum for the growth of the plants, the chemical compounds contain- 
ing the mineral matters necessary for plant growth being added to it, of 
course. Some of the advantages of this media are : it makes possible the 
most rigidly pure cultures ; the transparency of the agar permits the roots 
to be at all times visible ; contaminations are easily recognized ; and it af- 
fords a good mechanical support to the plants. The medium requires no 
attention beyond the initial preparation, that is, if a sufficient amount of 
medium is used at the beginning it does not require to be restored or re- 
newed, even during a long period. This greatly lessens the danger of 
contamination. A 1-per cent agar solution was employed. This con- 
tained relatively little inert material, and sufficient water to last several 
months. The roots of the plants grew largely on the outside of the jelly- 
like, agar mass, which, as it gradually shrank away from the walls of the 
vessel, allowed good aeration of the roots. Agar vv^as first employed as a 
culture substratum for green plants by Harrison and Barlow (14), who 
made use of it in their experiments with leguminosae. In the culture 
flasks in the experiment here recorded many of the plants grew well until 
all the water in the medium had been absorbed and the agar was dried 
down to a very small mass around the roots. 

The agar medium was used in all of the experiments herein described, 
after the first preliminary ones. The roots of the plants were grown un- 
der sterile or inoculated conditions and the upper part exposed to the air. 
In order successfully to secure these conditions, some suitable culture 
jars had to be provided. For this purpose Erlenmeyer flasks of Jena, 
Resistenz or Bohemian glass of 700 and 1000-c.c. capacity were used. 
These were nearly filled with agar medium and sterilized in the autoclave 
for 20 minutes at 12 to 15 pounds pressure. Following the methods of 
Hutchinson and Miller (16), Schulow (48) and Combes (11) cotton 
plugs were placed in the mouths of the flasks, each plug rolled around a 
glass tube about 1 cm. in diameter and 15 cm. in length, through which 
the young plant could grow. This tube was also plugged with cotton. 
When the top was reached by the plant, the tube was withdrawn and the 
cotton pressed about the plant. This method allowed free growth of the 
leaves in the air, and aeration as well as sterile condition of the roots. 



164 SOIL SCIENCE 

where this was desired. Each culture flask was wrapped with black 
paper to exclude light from the roots. 

The experiments described here were carried out with two varieties of 
com, namely, Zea Mays everta, Sturtevant (pop corn) ; and Zea Mays 
indentata, Sturt. (dent corn). 

The pop corn seedlings were all grown in the greenhouse the first year, 
but the dent corn seedlings the second year were grown in the large south 
windows (9 feet high and 12 feet wide) of the laboratories of the Science 
Building, because the new botanical greenhouses were not completed and 
the old one was unavailable. The air of the rooms was kept moist by 
sprinkling the floors and having large pans of water exposed in the 
room. The cultures were maintained for two to three months. 

Two methods were employed for measuring the growth of the plants. 
During the growth at various intervals and at tlie completion of the ex- 
periment the leaves were measured, and with the dent corn the dry weight 
of both the tops and the roots was obtained. The measurements were 
made first from the seed to the top of the youngest leaf and to this was 
added the length of each leaf from the stalk to its tip. The complete mea- 
surement of the plant was recorded. The measurements at intervals dur- 
ing growth did not reveal any special characteristics so they are not re- 
corded in the data presented in this paper. The dry weights of the 
whole plant were determined after the removal of the remains of the seed. 
The roots were freed from the agar which remained about tliem by melt- 
ing the agar in the autoclave and then washing the roots in boiling water. 
All of the substances occurring in the roots which are soluble in hot 
water were, of course, lost during this treatment. Each plant was then 
placed in a separate envelope and dried at a temperature of 80° C. to con- 
stant weight. These data of measurements and weights allow for accur- 
ate comparison with the checks which were grown in each series. 

The results were recorded and studied in three different forms : by 
means of the tables compiled from the figures obtained and recorded later 
in this paper ; by a comparison of photographs taken of the different sets 
grown at different times; and by graphs drawn for an easier and more 
ready comparison. Inoculated and sterile cultures were compared. 

Twelve cultures were prepared in a set, each containing the same 
nitrogen source. Six of each set of 12 cultures were sterile and 6 were 
inoculated. One healthy seedling was planted in each flask. For the first 
series, the flasks were inoculated with 10 drops of soil water, prepared by 
shaking 5 gm. of soil with 50 c.c. of distilled water. In all of the other 
series where the flasks were inoculated, a pure culture of B. subtilis was 
used. A loop of bacteria was transferred with a sterile platinum wire 
loop from an agar slant to the warm liquid agar culture flask and thor- 
oughly distributed through the medium by stirring with the sterile needle 
and shaking. In every case the inoculated flask showed a good growth of 
the bacteria. 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 165 

Seed Sterilization 

At the beginning- of the work the seeds were steriHzed by immersing 
in a water solution of mercuric chloride, 1:500, for 20 minutes. The 
seeds were first immersed in alcohol to remove any film of air. After the 
mercuric chloride treatment they were rinsed in sterile water to remove 
the sterilizing agent. This method was successful for the dent corn ; but 
when the pop corn was so treated only a poor germination was obtained, 
and weak seedlings resulted from the few seeds that did germinate. 

These results made it necessary to^ employ some other method for 
sterilizing pop corn, and following the suggestion of Lipman and Fowler 
(28), sulfuric acid (1.84 specific gravity) was tried. The best results 
were obtained by immersing the seeds for 4 minutes and then rinsing them 
in sterile water. 

The sterilized seeds were placed on moist filter paper in sterile Petri 
dishes and allowed to germinate. In three or four days those that germin- 
ated well were transferred with sterile forceps to the surface of the agar 
medium in the flasks, and later the young shoots were directed into the 
glass tubes, w^hich reached above the cotton stoppers. Germinating the 
seeds on agar was tried but the surface was too dry for the best results. 
Great care was used in selecting the seedlings to have them as nearly alike 
as possible, yet in spite of this precaution there was considerable differ- 
ence in the rapidity of the growth during the first two weeks. Some that 
appeared healthy would not reach the tops of the tubes for a week or 
more after others which seemed equally as good. This difference is one 
of the greatest sources of error in the method used but its effect is mini- 
mized by the use of large numbers of plants. 

Nutrient Solutions 

The nutrient solution in these cultures was one which has been found 
to be most successful by Professor Pollock, after extensive experiments 
in his laboratory. The tribasic calcium phosphate was used instead of the 
acid phosphate to assure an alkaline medium. This has low solubility but 
by using an excess of the phosphate the solution was constantly kept sup- 
plied witli a quantity sufficient for the growth of the plants. The amoimt 
of the different organic nitrogenous compounds to be used in the various 
solutions was determined upon the basis of furnishing in each solution the 
same amount of nitrogen that was present in the .004 M. solution of sod- 
ium nitrate used. Since peptone does not have a definite chemical for- 
mula, the nitrogen could not be accurately calculated, but it was esti- 
mated that 0.2 gm. of peptone in a liter of water would give the required 
amount of nitrogen. 

A stock solution was used for the check and to this was added single 
nitrogenous substances in the preparation of the other media. The follow- 
ing is a list of the substances used in the stock solution^ together with the 



166 SOIL SCIENCE 

number of grams per liter of water of each substance used : calcium phos- 
phate (tribasic) 1.240; magnesium sulfate 0.246; potassium chloride 
0.298; ferric chloride 5 c.c. of .001 M. solution. This solution furnishes 
all of the elements necessary for the growth of green plants except nitro- 
gen, and those obtained from water and carbon dioxide. 

The other culture media were made up by adding each of the following 
substances to the stock solution (the number of grams of each used per 
liter of stock solution is indicated) : sodium nitrate 0.340; urea 0.120 
peptone 0.2; guanin 0.120; guanidin carbonate 0.180; benzamid 0.484 
caffein 0.194; alanin 0.364; ammonium sulfate 0.264; asparagin 0.264 
glycocoll 0.300; uric acid 0.168; diphenylamin 0.676; guanidin nitrate 
0.122; hemoglobin 0.634; casein 0.459; linseed meal 1.120; cottonseed 
meal 1.090; malt 1.596; creatin 0.174. 

With the exceptions of cottonseed meal, malt, peptone and linseed meal, 
all of the substances used in these nutrient solutions were chemically pure, 
and distilled water from the chemical laboratory was used in all cultures. 
The organic nitrogenous compounds employed were those prepared by C. 
A. F. Kahlbaum. The cottonseed meal and linseed meal were secured 
from a retail feed store, and the malt which was obtained from a brewery 
consisted of ground, sprouted barley grains. 

Experiments 
Pop Corn 

Series I 

The plants of this series were started the middle of October, 1914, 
and were harvested the middle of March, 1915. They were grown in the 
greenhouse in the following media : check consisting of the stock solution ; 
and separate sets of media compound of stock solution to which sodium 
nitrate, urea, peptone, guanin and guanidin carbonate were respectively 
added. Half of the flasks were kept sterile while the other half were in- 
oculated with soil water, as has been described above. 

Soil water was not again used for inoculation because of bad results. 
The addition of this mixed culture of bacteria and fungi from the soil 
included some parasitic forms, which were detrimental to the plants. 
Therefore, in the succeeding series a pure culture of B. suhtilis was used. 
The results of this series are incorporated in Tables I and II. 

Series II 
The cultures of Series II were started February, 1915, and harvested 
in June of the same year. They were grown in the greenhouse, and the 
same media were used as in Series I. The flasks which were here inocu- 
lated, had pure cultures of B. suhtilis added, as has been described in the 
section on methods and technique. The plants of this series made a better 
and more uniform growth than those of Series I. Some of the plants 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 

TABLE I 

SERIES I, POP CORN, STERILE CULTURES 
(October, 1914 — March, 1915) 



167 



Culture 


Number 

of 

plants 


Total length 

of leaves 

cm. 


Average length 

of leaves 

cm. 


Per cent of 

average length of 

leaves of check 


Check 

Sodium Nitrate 

Urea 

Peptone 


4 

4 

3 

4 

2 


255 
200 
ISO 
145 
425 
365 
335 
245 
300 
290 
165 
335 
315 
225 
220 
180 
115 


187.50 

342.50 

251.70 
273.75 

147.50 


100.0 
182.6 

134.2 

146.0 

78.6 


Guanidin Carbonate*.. 





Plants small and died within a few days. 



TABLE II 

SERIES I, POP CORN, INOCULATED WITH SOIL WATER 
(October, 1914 — March, 1915) 





Number 


Total length 


Average length 


Per cent of 


Culture 


of 


of leaves 


of leaves 


average length of 




plants 


cm. 


cm. 


leaves of check 


Check 


5 


320 
275 
250 
190 
170 


241.00 


100.0 


Sodium Nitrate 


4 


280 
260 
230 
220 


247.50 


127.6 


Urea 


4 


575 
375 
330 
300 


395.00 


163.9 


Peptone 


4 


230 


211.25 


87.6 






220 










215 










180 






Guanin 


3 


320 
290 
280 


303.00 


125.8 


Guanidin Carbonate*. . 











* Plants small and died within a few days. 



168 



SOIL SCIENCE 



showed contaminations which were parasitic. These were discarded from 
the data, and this was done in ail of the following series. The contamina- 
tions in tlie flasks may have occurred on the seeds, some bacteria or fungi 
havii-^g survived the seed sterilization, or they may have gained entrance at 
the time of planting the seeds, when it was necessary to open the flasks. 
The results obtained here are similar to those of Series I, and may be 
studied by referring to Tables III and IV, 

TABLE III 

SERIES II, POP CORN, STERILE CULTURES 

(February-June, 1915) 





Number 


Total length 


Average length 


Per cent of 


Culture 


of 


of leaves 


of leaves 


average length of 




plants 


cm. 


cm. 


leaves of check 


Check 


12 


290 


235 


100.0 






285 








275 










265 










250 










230 










230 










210 










205 










200 










195 










185 






Sodium Nitrate 


7 


450 
445 
405 
370 
355 
335 
325 


384 


163.4 


Urea 


8 


370 
360 
355 
295 
280 
270 
230 
230 


299 


127.2 


Peptone 


10 


325 


253 


107.6 






305 










290 










275 










250 










230 










230 










200 






Guanin* 






' 




Guanidin Carbonate* . . 





* Plants small, no roots, and soon died. 

Series III 
The plants in Series III were started the last of June, 1915, and har- 
vested in about 8 weeks, the growth being very rapid during the long days 
and intense heat of the summer months. The greenhouse had rather 
poor means of ventilation and the glass was not painted so that the tern- 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 



169 



perature often reached 52° C, but the plants survived and made fairly 
good growth. The following nitrogen compounds were used in this set in 
addition to the check : sodium nitrate, urea, and peptone. The results are 
similar to those of the preceding series, and are recorded in Tables V and 
VI. 

TABLE IV 

SERIES II, POP CORN, INOCULATED WITH B. SUBTILIS 

(February-June, 1915) 





Number 


Total length 


Average length 


Per cent of 


Culture 


of 


of leaves 


of leaves 


average length of 




plants 


cm. 


cm. 


leaves of check 


Check 


11 


365 
320 


246 


100.0 










315 










290 










230 










225 










225 










220 










190 










165 










155 






Sodium Nitrate 


7 


610 
530 
400 
400 
310 
300 
■300 


407 


165.4 


Urea 


11 


400 
390 
380 
375 
370 
370 
370 
365 
360 
355 
335 


370 


150.4 


Peptone 


10 


440 
420 
360 
320 
315 
315 
310 
305 
265 
250 


330 


134.1 


Guanin* 










Guanidin Carbonate* . . 











* Plants small, no roots, and soon died. 



The study of the data upon these first three series revealed very similar 
results in all. A summary of these results is shown in Table VII and 
figure 1. Because of the large number of plants grown, some definite con- 
clusions may be drawn from the experiments, which will be stated later. 



170 



SOIL SCIENCE 



Series IV 
The plants of this series were grown at the same time as, and under 
similar conditions to those of Series III, except that water cultures were 
used instead of agar cultures. There was no provision made for aeration. 

TABLE V 

SERIES III, POP CORN, STERILE CULTURES 

(June- August, 1915) 





Number 


Total length 


Average length 


Percent of 


Culture 


of 


of leaves 


of leaves 


average length of 




plants 


cm. 


cm. 


leaves of check 


Check 


5 


290 

210 
190 
180 
180 


210 


100.0 


Sodium Nitrate 


3 


320 
300 
220 


283 


134.7 


Urea 


3 


180 
178 


178 


84.7 










175 






Peptone ^ 


5 


420 
370 


313 


149.0 










300 










1245 










230 







In addition to the check, media containing the following nitrogen com- 
pounds were used : sodium nitrate, urea, peptone, benzamid, caffein, 
alanin, ammonium sulfate, and asparagin. By consulting Tables VIII 

TABLE VI 

SERIES III, POP CORN, INOCULATED WITH B. SUBTILIS 

(June-August, 1915) 



Culture 


Number 

of 

plants 


Total length 

of leaves 

cm. 


Average length 

of leaves 

cm. 


Per cent of 

average length of 

leaves of check 


Check 


6 

4 
4 
2 


320 
260 
250 
230 
195 
190 
430 
410 
310 
170 
585 
430 
325 
280 
340 
260 


224 

340 

405 
300 


100.0 


Sodium Nitrate 

Urea 


151.7 
180.8 




133.9 







and IX and comparing the growth with that in the agar medium, it can 
readily be seen that the growth in these water cultures was exceedingly 
poor in all cases, and not nearly equal to that in the agar cultures. The 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 



171 



TABLE VII 

SUMMARY OF DATA OF LENGTHS OF LEAVES OF 110 POP CORN PLANTS GROWN 

IN DIFFERENT CULTURE MEDIA, UNDER STERILE CONDITIONS AND 

INOCULATED WITH B. SUBTILIS, 1914-1915 





Sterile Cultures 


Inoc. B. subtilis 


Culture 


Average 

length 

cm. 


Per cent of 

average length 

of check 


Average 

length 

cm. 


Per cent of 

average length 

of check 


Check 

Sodium Nitrate. . 

Urea 

Peptone 

Guanin* 

Guanidin 

Carbonate* . . . 


227.7 
353.5 
265.7 
272.3 


100.0 
155.2 
116.6 
119.6 


243.9 
379.0 
379.3 
325.0 


100.0 
159.4 
159.5 
133.2 



Toxic, no growth. 







POP 


CORI 


M 










cm. 

400 


















300 


\ 


^-^. 


s 






*§*>»» (w! B -» ^ ' ■ 


tarea 


—_ 


Inoc. 


B.5d 


zoo 


\ 
















100 


















000 






























^ 










o 


1 ft 














Z5 






o 


zS 









Fig. 1. — A summary of the results obtained from all of the pop corn plants grown 
in the experiment. 



172 



SOIL SCIENCE 



plants were weak and sickly. It was decided therefore, that unaerated 
water cultures were unsuitable for these experiments, and thereafter only 
agar cultures were used. The relative value of the nitrogenous substances 
in the water cultures was similar to that of these substances in the agar 
cultures, and may be used in connection with them. 

TABLE VIII 

SERIES IV, POP CORN, WATER CULTURES, STERILE CONDITIONS 

(June-August, 1915) 





Number 


Total length 


Average length 


Per cent of 


Culture 


of 


of leaves 


of leaves 


average length of 




plants 


cm. 


cm. 


leaves of check 


Check 


4 


160 
120 
110 
100 


123.0 


100.0 


Sodium Nitrate 


5 


310 
240 
240 
190 
160 


228.0 


185.3 


Urea 


3 


165 
160 


160.0 


130.0 










155 






Peptone 


4 


230 
210 
120 


170.0 


138.0 


Benzamid* 


2 


120 
160 


140.0 




Caffein 


113.8 






120 






Alanin ^ 


4 


140 
140 


127.5 


103.6 










120 










110 






Ammonium Sulfate . . > 


3 


160 
120 

120 


133.3 


108.3 


Asparagin 


5 


225 
120 


135.0 


109.8 










115 










110 










110 







* Toxic, no growth. 

Dent Corn 
In the preceeding experiments some difficulty had been found in ob- 
taining pop com seedlings, and because of this fact and because it was ad- 
visable to try the effect of these substances upon another variety of the 
species, dent corn was used. Also other chemical substances were used 
as sources of nitrogen. 

Series V 
The plants of Series V were started in October, 1915, and harvested 
the following February. The plants were grown in the south window of 
one of the botanical laboratories. The light was not as good here as in 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 



173 



the greenhouse, but fairly uniform growth was obtained. The following 
nitrogen compounds were tested : sodium nitrate, urea, peptone, guanin, 
guanidin cabornate, guanidin nitrate, benzamid, caffein, alanin, ammon- 
ium sulfate, asparagin, glycocoll, uric acid, diphenylamine. In these, as in 
all the experiments, plants were grown in the stock solution as a check. 

The results of these experiments are given in Tables X and XI. It is 
interesting to compare these results with those found in the growth of the 
pop corn plants. In general they are similar, but guanin which was toxic 
to pop corn was found to be quite beneficial to the dent corn seedlings. 

TABLE IX 
SERIES IV, POP CORN, WATER CULTURES, INOCULATED WITH B. SUBTILIS 

(June- August, 1915) 





Number 


Total length 


Average length 


Per cent of 


Culture 


of 


of leaves 


of leaves 


average length of 




plants 


cm. 


cm. 


leaves of check 


Check 


5 


140 
135 
135 
120 
100 


126 


100.0 


Sodium Nitrate 


5 


185 
175 
170 
170 
150 
150 


166 


131.7 


Urea 


1 


120 


120 


95.2 






Benzamidt 










Caffein 


S 


150 

115 

110 

95 

90 


112 


88.8 


Alanin , 


2 


130 
110 


120 


95.2 


Ammonium Sulfate . . , 


5 


250 
160 

90 
85 
80 


133 


105.5 


Asparagin 


1 


140 


140 


119.0 



No plants obtained. 



t Toxic, no growth. 



Series VI 
The plants of this series were started November, 1915, and the growth 
terminated during the next March. They were grown in a very poorly 
lighted window of one of the botanical laboratories, and consequently the 
growth was poor and very irregular. These facts must be considered in 
drawing any conclusion from the results of this series. The following 
nitrogenous substances were used : hemoglobin, casein, linseed meal, cot- 
tonseed meal, and malt. The results may be studied in Tables XII and 
XIII. 



174 



SOIL SCIENCE 



TABLE X 

SERIES V, DENT CORN, STERILE CULTURES 

(October, 1915~February, 1916) 





Number 


Totallgth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of « 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt. 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Check 


5 


285 
250 
230 
180 
150 


219 


0.42 
0.28 
0.30 
0.25 
0.25 


0.30 


100.0 


Sodium Nitrate 


4 


410 
325 
280 
270 


321 


1.08 
0.62 
0.63 
0.52 


0.71 


236.6 


Urea 


4 


320 
180 
160 
110 


182 


0.75 
0.30 
0.22 
0.06 


0.33 


110.0 


Peptone 


4 


345 


296 


1.18 


0.66 


220.0 






345 




0.60 










260 




0.50 










235 




0.35 






Guanin 


5 


404 
385 
365 
350 
250 


351 


1.24 
1.40 
1.10 
1.30 
0.46 


1.10 


366.6 


Guanidin Carbonate . 


4 


165 
155 
120 
105 


138 


0.35 
0.30 
0.15 
0.20 


0.25 


83.3 


Benzamid* 














Caffein 


6 


150 
145 
135 
110 
80 
25 


109 


0.20 
0.31 
0.21 
0.20 
0.13 
0.10 


0.19 


63.3 


Alanin 


6 


420 
365 
320 
270 
225 
215 


300 


1.90 
1.28 
1.05 
0.51 
0.47 
0.44 


.94 


313.3 


Ammonium Sulfate. . . 


3 


300 
290 
235 


275 


0.65 
0.62 
0.35 


.54 


180.0 


Asparafifin ....••>■•• 


5 


365 


319 


1.40 


.84 


290.0 






335 




0.94 










315 




0.71 










310 




0.70 










270 




0.45 






Glycocoll 


5 


365 


281 


1.14 


.63 


210.0 






325 




0.70 










245 




0.52 










240 




0.40 










230 




0.40 






Uric Acid • ...• 


6 


280 
270 


256 


0.48 
0.47 


.44 


146.6 










265 




0.57 










265 




0.38 










260 




0.45 










200 




0.31 







BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 

TABLE X— Continued 



175 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Percent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry vifeight 


av. dry wgt. 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Diphenylamin* 














Guanidin Nitrate 


4 


160 
135 
130 
110 


134 


0.31 
0.27 

0.22 
0.15 


.24 


80.0 



* No growth, died within three days. 

Series VII 

This was one of the most successful sets grown during the year. The 
plants were started in January, 1916, and harvested in about three 
months. They were grown in a well lighted window of one of the labora- 
tories of the Science Building, and the room was well heated. The fol- 
lowing media were used : distilled water, check, sodium nitrate, urea, pep- 
tone, guanin, alanin, ammonium sulfate, asparagin, uric acid, hemoglobin, 
casein, linseed meal, cottonseed meal, malt, and creatin. 

The plants of this series all made good growth and some interesting 
results were obtained, which may be readily seen by a study of Tables 
XIV and XV. The results of this and of the other series are discussed 
later in this paper. 

Series VIII 
The plants of this series were started in February, 1916, and harvested 
about two months later. They were grown in large test tvibes. These 
plants had only about half the amount of medium that the plants of other 
series had, and consequently the growth had to be terminated at an earlier 
stage, but nevertheless, interesting results were obtained. The plants 
were grown in the new botanical greenhouse under very ideal conditions 
of light and heat, and a very good and uniform growth resulted. The 
following substances were used : check, sodium nitrate, urea, uric acid, 
casein, and cottonseed meal. The detailed results of this series are given 
in Tables XVI and XVII. 

Discussion 

The data presented in this thesis, comprise observations upon 614 
Zea Mays plants grown until the water supply became exhausted in one 
or more of the culture flasks of a series. The conclusions are based upon 
the results of growth of these plants. This number does not include 
those plants which showed extreme toxic effects when young and made 
no further growth, nor those discarded because they were attacked by 
fungi. The large number of the plants employed makes it possible for 
us to draw certain fairly definite conclusions from the data secured. 

The percentage of possible error in such work is a large one and must 
be taken into account in interpreting the result obtained. There are sev- 



176 



SOIL SCIENCE 



TABLE XI 

SERIES V, DENT CORN, INOCULATED WITH B. SUBTILIS 

(October, 1915— February, 1916) 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Check 


6 


215 
200 
190 
175 
170 
135 


181 


0.30 
0.28 
0.30 
0.30 
0.20 
0.12 


0.25 


100.0 


Sodium Nitrate 


4 


340 
270 
250 
200 


265 


0.65 
0.45 
0.38 
0.25 


0.40 


160.0 


U rea 


5 


345 


231 


0.60 


0.30 


156.0 






370 




0.70 






165 




0.28 










160 




0.16 










145 




0.20 






Peptone 


4 


375 


345 


1.00 


0.96 


384.0 






350 




0.93 










330 




0.70 










325 




1.20 






Guanin 


6 


400 
335 
320 
275 
270 
270 


311 


1.07 
1.04 
0.47 
0.60 
0.70 
0.50 


0.73 


292.0 


Guanidin Carbonate. . 


4 


210 
175 
170 
130 


171 


0.44 
0.30 
0.25 

0.25 


0.31 


124.0 


Benzamid* 


6 


210 


130 


0.34 


0.23 




Caffein 


92.0 






170 




0.23 










140 




0.22 










115 




0.22 










80 




0.20 










55 




0.15 






Alanin 


6 


430 
330 


300 


1.20 
1.50 


0.88 


352.0 










330 




1.05 










270 
255 
185 




0.55 
0.69 
0.32 






Ammonium Sulfate . . 


5 


430 
345 
325 
300 
300 


340 


1.52 
0.97 
0.75 
0.65 

0.54 


0.85 


348.0 


Asparagin 


5 


420 
400 
300 
285 
275 


336 


0.90 
1.34 
0.69 
0.68 
0.57 


0.83 


332.0 


Glycocoll 


5 


325 
225 
220 
220 
200 


240 


0.82 
0.40 
0.67 
0.53 
0.37 


0.56 


224.0 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 



177 







TABLE 


XI — Continued 








Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 




5 


415 
320 


230 


1.02 
0.50 


0.48 


192.0 










215 




0.32 










170 




0.29 










170 




0.27 






Diphenylamin* 














Guanidin Nitrate .... 


3 


150 
130 


135 


0.26 
0.22 


0.22 


88.0 






125 




0.20 







* No growth, died within three days. 

eral sources of error of which the most serious is the individual differ- 
ences which occur between plants. No two individuals are exactly alike, 
as is shown, for instance, by the different growth vigor of different plants 
under identical external conditions. The degree of this error diminishes 
with increase in the number of plants. A second factor is the light rela- 
tion. In the climate of southern Michigan, during the winter months on 

TABLE XII 

SERIES VI, DENT CORN, STERILE CULTURES 

(November, 1915— March, 1916) 





Number 


Total Igth, 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Check 


6 


330 
320 
300 
300 
285 
220 


289 


1.25 
1.52 
1.22 
0.90 
1.00 
0.55 


1.07 


100.0 


Hemoglobin 


5 


335 
335 
280 
255 
210 


283 


2.20 
0.55 
1.34 
0.60 
0.40 


1.02 


95.3 


Casein 


4 


400 
255 
250 
230 


284 


3.00 
1.00 
0.50 
1.35 


1.46 


130.4 


Linseed Meal 


5 


355 
330 
315 
285 
280 


313 


2.26 
1.70 
1.05 
1.30 
1.00 


1.46 


136.4 


Cottonseed Meal 


5 


405 
370 
350 
310 
255 


338 


1.34 
2.20 
1.65 
1.67 
1.55 


1.68 


157.0 


Malt 


6 


390 
380 


328 


1.25 
0.95 


1.13 


105.6 










335 




2.35 










300 




0.85 










300 




0.75 










265 




0.65 







(iii— 14) 



178 



SOIL SCIENCE 



account of the shorter days, less intensity of the sunlight and the large 
proportion of the cloudy days, the light at the disposal of the plants is much 
less than during the summer months. As a result, the rate of growth is 
less than in summer. This fact must be taken into consideration when 
comparing the growth of series which were grown at different times of 
the year. Also, those plants which were grown in the laboratory win- 
dows did not receive as much light as those in the greenhouse, and those 
standing near the windows received more than plants farther back. This 

TABLE XIII 

SERIES VI, DENT CORN, INOCULATED V/ITH B. SUBTILIS 

(November, 1915 — March, 1916) 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Check 


5 


360 
335 
325 
310 
255 


316 


1.65 
I.IS 
1.40 
0.70 
1.40 


1.22 


100.0 


Hemoglobin 


4 


350 
260 
260 
185 


264 


0.60 
1.30 
0.85 

0.35 


0.78 


62.3 


Casein 


3 


400 
300 
210 


303 


2.70 
1.42 
0.50 


1.54 


126.2 


Linseed Meal 


4 


370 
365 
315 
280 


332 


2.82 
1.30 
0.90 
2.19 


1.61 


123.7 


Cottonseed Meal .... 


6 


365 
310 
300 
280 
255 
230 


290 


2.98 
1.50 
1.70 
1.17 
1.25 
1.05 


1.61 


123.7 


Malt 


S 


355 

345 
340 
320 
235 


310 


0.90 
1.45 
1.25 
0.90 
0.56 


1.01 


82.7 



was controlled by shifting their positions during the period of growth. 
The diminishing of light causes a lessening of carbohydrate production 
and hence slower growth. This slow development may somewhat influ- 
ence the assimilation of nitrogen. Another possible source of error is the 
wide temperature variations which occurred while some of the series were 
being grown. During vacations the heat in the building where the plants 
were grown was reduced and this caused a check in growth in Series V 
and VI from which the plants did not fully recover. These factors, then, 
which influence the percentage of error must be born in mind when mak- 
ing comparisons between different series. The large error due to differ- 
ences in individual plants is well illustrated in the check solution of Series 



BRIGHAM-ASSIMILATION OF ORGANIC NITROGEN 179 



TABLE XIV 

SERIES VII, DENT CORN, STERILE CULTURES 
(January-March, 1916) 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Distilled Water 


3 


275 
215 
170 


220 


0.93 
0.55 
0.40 


0.62 


47.3 


Check 


6 


375 
360 
345 
330 
305 
230 


324 


1.65 
1.20 
1.30 
1.20 
1.41 
1.15 


1.31 


100.0 


Sodium Nitrate 


4 


400 
385 
345 
290 


370 


2.75 
2.60 
1.90 
1.42 


2.17 


185.6 


Urea 


5 


465 


375 


3.12 


2.00 






152.6 






430 




2.40 










390 




1.73 










320 




1.50 










270 




1.27 






Peptone 


5 


435 


353 


2.47 


1.78 


135.8 








365 




1.47 










350 




2.60 










310 




1.05 










305 




1.30 






Guanin 


6 


400 
360 
360 
350 
345 
275 


348 


2.15 
1.90 
1.90 
1.20 
1.42 
1.05 


1.60 


122.1 


Alanin 


4 


335 


291 


2.00 


1.50 


114.5 








300 




2.45 










280 




0.90 










250 




0.67 






Ammonium Sulfate. . . 


6 


495 
485 
460 
400 
360 
350 


425 


2.70 
3.85 
3.30 
2.02 
1.70 
1.12 


2.45 


187.0 


Asparagin 


6 


560 


523 


5.10 
4.25 


3.89 


296.9 






550 






550 




4.20 










510 




2.77 










500 




4.10 










470 




2.97 






Uric Acid 


4 


560 
520 
435 
400 


478 


3.85 
3.35 
2.47 
2.98 


3.16 


241.2 


Hemoglobin 


6 


485 
425 
365 
325 
300 
285 


364 


3.75 
2.60 
1.42 
1.20 
1.52 
1.02 


1.92 


146.5 



180 



SOIL SCIENCE 

TABLE XIV— Continued 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt. 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Casein 


6 


490 
460 
440 
420 
400 
320 


422 


3.65 
2.54 
2.80 
3.00 
2.70 
1.25 


2.68 


204.5 


Linseed Meal 


5 


415 
335 
310 
300 

275 


327 


2.75 
1.35 
0.97 
1.30 
1.50 


1.57 


112.2 


Cottonseed Meal 


6 


410 
400 
360 

280 
245 
230 


321 


2.90 
2.15 
2.10 
1.40 
1.07 
1.37 


1.83 


139.6 


Malt 


6 


330 
325 
320 
310 
275 
250 


301 


1.25 
1.25 
1.47 
0.97 
0.77 
0.65 


1.05 


80.1 


Creatin 


6 


400 
360 
355 
350 
345 
275 


347 


2.20 
1.45 
1.65 
1.40 
1.45 
1.37 


1.58 


120.6 



II. The largest plant measured 365 cm. and the smallest 155 cm., a dif- 
ference of 210 cm. However, in all the checks of all the series, 48 
plants in sterile cultures averaged 227.7 cm., and 49 plants in inoculated 
cultures averaged 221.6 cm. a difference of only 6.1 cm. With this num- 
ber of plants the margin of error is very small. 

The means for determining the amount of development of the plants 
in the various compounds used was, as has been stated above, by measure- 
ment of the length of the stalks and leaves, and by determining the dry 
weight. A comparison of the data obtained by the two methods shows 
that they are nearly parallel. The data show that in 19 cases the measure- 
ments and weights are, respectively, in the same relation in the sterile and 
the inoculated cultures ; but in 5 cases they are reversed. This may be 
partly explained by the fact that the cultures in which these reverses oc- 
curred were checked in their growth, as has been explained. The leaves 
then did not develop well, but ears were formed which increased the 
weight. The weights probably serve a more definite and accurate basis 
for comparison than the measurements (cf. Tables X-XVII). 

Since the problem was to determine the availability of various organic 
nitrogenous compounds for higher plants, the most logical means of dis- 
cussion seems to be to take up each compound separately, explain the re- 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 



181 



TABLE XV 

SERIES VII, DENT CORN, INOCULATED WITH B. SUBTILIS 
(January-March, 1916) 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt. 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Distilled Water 


2 


220 
140 


180 


0.72 
0.28 


0.50 


34.5 


Check 


S 


350 
330 
325 
265 
215 


297 


1.79 
1.68 
1.65 
1.20 
0.95 


1.45 


100.0 


Sodium Nitrate 


4 


410 
370 
320 
300 


350 


2.32 
2.80 
0.80 

0.70 


1.65 


113.8 


Urea 


S 


480 
465 
405 
355 
225 


350 


2.35 
3.95 
1.67 
1.65 
0.70 


2.10 


144.8 


Peptone 


6 


490 
440 


435 


3.45 
2.32 


2.35 


162.0 










440 




1.57 










430 




3.00 










430 




1.70 










380 




2.10 






Guanin 


6 


495 
490 
470 
465 
420 
385 


454 


3.55 
3.10 
2.25 
2.35 
1.92 
1.97 


2.52 


173.8 


Alanin 


6 


400 
400 
315 
305 
300 
280 


333 


2.97 
2.95 
2.60 
1.30 
1.40 
1.38 


2.10 


144 8 


Ammonium Sulfate. . . 


6 


510 
465 
465 
460 
445 
430 


462 


3.45 
3.57 
3.17 
2.17 
3.15 
3.41 


3.15 


217.2 


Asparagin 


4 


610 
600 

535 
500 


561 


4.69 
4.55 
3.77 
3.95 


4.24 


292.4 


Uric Acid 


6 


480 
475 


417 


3.32 
2.52 


2.51 


173.1 










460 




3.55 










400 




3.12 










390 




1.60 










300 




0.97 






Hemosrlobin 


6 


550 


502 


4.30 


3.61 


248.0 






540 




3.15 










535 




4.27 




• 






510 




3.80 










505 




3.65 










375 




2.49 







ia2 



SOIL SCIENCE 

TABLE XV— Continued 





Number 


Totallgth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Casein 


4 


485 
470 


464 


4.85 
3.42 


3.79 


261.4 










450 




3.57 










450 




3.35 






Linseed Meal 


6 


425 
375 
350 
350 
330 
330 


360 


3.10 
1.65 
1.95 
1.32 
2.85 
1.60 


2.08 


143.4 


Cottonseed Meal .... 


6 


460 
400 
370 
370 
360 
315 


380 


3.20 
2.82 
2.20 
2.12 
2.52 
2.70 


2.59 


178.6 


Malt 


6 


380 


331 


1.68 
1.55 


1.50 


103.4 


1 




375 








370 




1.75 










360 




1.92 










270 




1.10 










230 




1.02 






Creatin 


6 


390 


340 


1 .60 


1.44 


99.3 






360 




1.45 






340 




1.20 










325 




1.72 










320 




1.17 










300 




1.50 







suits obtained in the different cultures, and show the significance which 
they seem to reveal. Therefore, this procedure has been adopted. 

Checks 

The check solution was used in all the series. It contained all the 
chemical elements necessary for the growth of plants except nitrogen and 
those which the plant gets from the air. The growth in this solution was 
taken as the amount of growth allowed by the nitrogen supply in the seed. 
In the culture solutions containing nitrogen a growth markedly less than 
that of the check was interpreted as a toxic effect. A growth equal to 
that of the check was assumed to indicate that the nitrogen was not avail- 
able. A growth markedly better than the check indicated that nitrogen 
in the form supplied was available. The plants grown in the check solu- 
tion, toward the end of the period of growth, always showed the yellow- 
ing of the leaves, a characteristic effect of the lack of nitrogen. 

The difference between the plants grown in the sterile cultures and in 
the inoculated ones is very slight. With the pop corn the plants inocu- 
lated in all cases were from 10 to 40 cm. better. Considering the mea- 
surement on length of all the check plants, those in the sterile cultures 
averaged 6 cm. per plant better than those in the inoculated. 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 



183 



Sodium Nitrate 
A complete nutrient solution containing sodium nitrate was employed 
in all the series but one. Since the time of Boussingault (8) sodium 
nitrate has been considered one of the best, if not the best, source of 
nitrogen. It is in common use as a commercial fertilizer. In Series II, 
III, and VIII of the experiments it produced the best growth of all sub- 
stances used. These were all grown in the greenhouse under favorable 

TABLE XVI 

SERIES VIII, DENT CORN, STERILE CULTURES 

(February-April, 1916) 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry wreight 


av. dry wgt 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Check 


6 


170 
170 
160 
155 
ISO 
140 


157.5 


1.52 
1.25 
1.10 
1.30 
1.25 
1.00 


1.23 


100.0 


Sodium Nitrate 


5 


215 
215 
205 
200 
185 


204.0 


1.90 
1.80 
1.72 
1.60 
1.80 


1.76 


143.0 


Urea 


3 


190 
190 
165 


181.6 


1.85 
1.70 
1.50 


1.68 


136.5 


Uric Acid 


6 


200 
190 
190 
185 
160 
150 


179.0 


1.80 
1.70 
l.SO 
1.50 
1.40 
1.45 


1.50 


126.8 


Casein 


6 


205 
200 
190 
175 
170 
170 


185.0 


1.65 
1.45 
1.70 
1.25 
1.25 
1.20 


1.41 


114.6 


Cottonseed Meal .... 


5 


160 

155 
145 
145 
135 


148.0 


1.28 
1.20 
1.10 
1.10 
1.02 


1.14 


92.6 



conditions of temperature and light. In Series VIII, the experiment was 
discontinued after only two months had elapsed because with the small 
amount of medium used the water was exhausted at the end of that 
period. 

Table VII and figure 1 show that in the growth of pop corn, sodium 
nitrate in sterile cultures was the best of the compounds tested as a source 
of nitrogen, while in the inoculated cultures urea equaled it in value. In 
the growth of dent corn the results in sterile cultures indicated that am- 
monium sulfate and asparagin are superior to sodium nitrate as a source 



184 



SOIL SCIENCE 



of nitrogen. In inoculated cultures, however, the following substances 
gave better results than the nitrate : asparagin, ammonium sulfate, pep- 
tone, guanin, uric acid, alanin, urea, hemoglobin, casein, linseed and cot- 
tonseed meals. The growth of the dent corn plants in the inoculated cul- 
tures of the nitrate was slightly poorer than in the sterile cultures, while 

TABLE XVII 

SERIES VIII, DENT CORN, INOCULATED WITH B. SUBTILIS 
(February-April, 1916) 





Number 


Total Igth. 


Avg. length 


Dry 


Average 


Per cent of 


Culture 


of 


of leaves 


of leaves 


weight 


dry weight 


av. dry wgt. 




Plants 


cm. 


cm. 


gm. 


gm. 


of check 


Check 


6 


155 
155 
140 
130 
125 
120 


137.5 


1.20 
1.15 
1.15 
0.80 
0.92 
0.95 


1.03 


100.0 


Sodiiun Nitrate 


6 


205 
205 
205 
195 
190 
170 


195.0 


2.15 
1.82 
1.85 
1.80 
2.10 
1.55 


1.88 


182.5 


Urea 


6 


180 
175 


166.0 


1.75 
1.88 


1.61 


156.3 










175 




1.35 










170 




1.90 










160 




1.90 










135 




0.90 






Uric Acid * 


5 


190 
190 


174.0 


1.80 
1.60 


1.57 


152.4 










180 




1.50 










170 




1.00 










140 




1.95 






Casein 


6 


215 
195 


185.0 


1.92 
1.47 


1.56 


151.4 










190 




1.73 










185 




1.67 










165 




1.27 










160 




1.32 






Cottonseed Meal .... 


6 


180 
180 
160 
155 
155 
135 


161.0 


1.45 
1.25 
1.30 
1.25 
1.10 
0.72 


1.18 


114.5 



in the pop corn plants the reverse was true, but the differences in both 
cases were within the range of error inherent in the method. 

From these experiments it is clear that in all cases the growth of 
plants when furnished sodium nitrate was markedly better than when no 
nitrogen, except that in the seed, was present. The poorest showing for 
the nitrate was 113.6 per cent of the check in Table XV; the best was 
236.6 per cent in Table X. 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 



185 



Urea 
The plants grown in cultures containing urea as the source of nitrogen 
showed in the case of the pop corn a decidedly better development than 
those in the check solution. This indicates that the nitrogen of urea is 
available to some extent, but not sufficiently to make urea equal to sod- 
ium nitrate. However, in the inoculated cultures it proved equal to the 
nitrate as a source of nitrogen. This indicates that amraonification or 
some other transformation of urea is necessary for the best utilization 
and assimilation of that compound by pop corn plants. The weight of the 
dent corn plants in the sterile cultures showed urea to be about 50 per 
cent better than the check, though the leaf measurements were no greater 







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Fig. 2. — A summary of all the dent corn plants grown, showing both the dry 
weights and the measurements. 

than those of the check. In the inoculated cultures both length and dry 
weight showed urea better than the inoculated check, and the dry weight 
showed even better growth than the dry weight of the plants in the in- 
oculated cultures of sodium nitrate. A comparison of the sterile and the 
inoculated cultures, containing urea, shows no difference in the dry weight 
and the length of leaf is only slightly better in the inoculated cultures. 

Urea was found unavailable by Pryanishnikov and Lyebyedyev (40), 
and toxic by Sawa (41). Hovv^ever, it has been reported beneficial by 
Hutchinson and Miller (16), Molhard (33), Suzuki (52) and Tompson 
(54). Takeuchi (53) has found that the enzyme, urease, which ammon- 
ifies urea, is not present in Mays. The com itself then cannot ammonify 
urea. 



186 SOIL SCIENCE 

Peptone 
Peptone was utilized by Mays plants in both sterile and inoculated 
cultures. In the sterile cultures in both varieties of corn it was better 
than urea but did not quite equal the nitrate as a source of nitrogen. As 
with urea, the action of B. subtilis seemed to increase its availability for 
Mays plants. With pop corn, in the inoculated cultures, the growtli was 
not equal to either that in the nitrate or urea but was considerably bet- 
ter than in the sterile cultures. With dent corn in sterile cultures the 
plants make a much better development than in the check but were not 
equal to those in the nitrate. The growth was 27 per cent better in the 
inoculated cultures than in the sterile. The plants with peptone were 
fifth in rank. Hutchinson and Miller (16) have found peptone a source 
of available nitrogen. 

Guanin 

Guanin was found to be exceedingly toxic to the pop corn plants in 
both sterile cultures and in those cultures which were inoculated with 
B. subtilis. These plants made practically no growth. In the cultures 
inoculated with soil water the growth was fair, but as there were only a 
few plants, no definite conclusions can be drawn. In the sterile cultures it 
was found to be about equal to the sodium nitrate for dent corn, and was 
better in the inoculated than in the sterile cultures. 

The results show clearly that as a source of nitrogen the same chemi- 
cal compound may have a value differing to a considerable degree for 
different varieties of a species. Guanin was toxic to pop corn and fur- 
nished available nitrogen to dent corn. Schreiner and Reed (44) and 
Schreiner and Skinner (46) have found guanin available to wheat seed- 
lings. 

Guanidin Carbonate 
In the experiments guanidin carbonate was found to be exceedingly 
toxic to the pop com plants used. Young seedlings made a very slight 
growth and died within a few days on culture media containing this sub- 
stance. It was less toxic to dent corn but none of the cultures with this 
substance were as good as the check. Guanidin carbonate has also been 
found quite toxic to other plants by Kawakita (18), Schreiner and Reed 
(44, 45), Schreiner and Skinner (46), and Bierma (4). 

Benzamid 
The plants grown in a nutrient solution containing benzamid showed 
a decidedly toxic effect of this substance. They made a very feeble 
growth and died within 3 weeks. The action of B. subtilis did not alter 
the toxicity of this substance. Lutz (30) found that all compounds con- 
taining the benzin ring group were toxic to plants. 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 1S7 

Caffein 
The caffein nutrient solution with dent corn was more toxic than the 
guanidin carbonate, and much poorer than the solution used in the check 
culture. The leaves of the plant were small and pale in color. Those 
plants grown in the inoculated cultures were slightly better than those in 
the sterile cultures but the difference was not very marked. This com- 
pound has also been reported toxic by Lutz (30). 

Glycocoll 
GlycocoU was used as the source of nitrogen in only one series of ex- 
periments. The results of this series showed that it was favorable to the 
growth of Mays plants. It did not prove equal to sodium nitrate in the 
single series in which it was used. The growth was very little better in 
the sterile cultures. Its effect on other plants has been ascertained and 
found favorable by Schreiner and Skinner (46), Hutchinson and Miller 
(16j, Dachnowski and Gormley (12), Lefevre (24), Molliard (34), and 
Schreiner and Reed (47). 

Uric Acid 
Thompson (54) has shown by his experiments that uric acid furnishes 
as good a source of nitrogen for oats as does urea and sodium nitrate. The 
results of the author's experiments with Mays are very similar. In the 
sterile cultures the growth of the dent corn was equal to that in the 
sodium nitrate both in length of leaves and in dry weight. Uric acid was 
better than urea as a source of nitrogen. There was only 1 cm. difference 
by measurement and .05 gm. by weight, between tlie averages of the 
sterile and the inoculated cultures in uric acid. 

Diphenylamin 
Diphenylamin was the most toxic substance used. When germinated 
seedlings were placed upon the agar medium containing this substance the 
roots turned brown and the plants died within 24 hours. 

Alanin 
The results of the experiments with alanin as a source of nitrogen, 
presented in Tables X, XI, XIV and XV, show it to be a good nitrogen 
source. In the sterile cultures the plants are nearly as good as those in 
the corresponding nitrate solution. The plants grown in the inoculated 
nitrate cultures are better than those of the sterile and better than the in- 
oculated nitrate cultures. It is, therefore, a good source of nitrogen for 
Mays, although Schreiner and Skinner (46) founds it slightly toxic to 
wheat seedlings and Molliard (34) reported it toxic to roots, while Le- 
fevre (24) found it favorable as a nitrogen source. 



188 SOIL SCIENCE 

Ammonium Sulfate 

Ammonium sulfate was used in Series V and VII of the experiments. 
It has been known from the time of Liebig to be very readily assimilated 
by some plants. In the experiments here reported it was found to give a 
better growth of Mays than most of the other substances tried, and much 
better than sodium nitrate or urea. The measurements of the leaves 
show the sterile cultures to be slightly better while the weights reverse 
the ratio. 

Asparagin 

Asparagin is a substance fotmd very widely distributed in plants, and 
the results obtained in these investigations show it to be an excellent 
source of nitrogen for Mays. The plants grown in this solution in sterile 
cultures are shown by measurements to be surpassed only by those in 
ammonium sulfate ; by weights they are far better than any others. The 
growth in inoculated cultures is about equal to that in sterile. It has also 
been found readily assimilated in the experiments of Baessler (2), Mol- 
liard (33, 34), Nakamura (37), and Skinner and Beattie (51). 

Guanidin Nitrate 
The effect of guanidin nitrate upon Mays v^ras about parallel to that 
of guanidin carbonate ; approximately the same growth was obtained, the 
former shovv^ing about the same toxic reaction as the latter. There was 
a difference in weight of onl}^ .02 gm. between the plants in the sterile 
cultures inoculated with B. subtilis. 

Hemoglobin 

Hemoglobin is a complex animal protein, and it might be expected 
that, due to the molecular structure, the nitrogen would not be available 
for plants. The results show that in the sterile cultures it was slightly 
better than the check both by measurements and weights, but not as good 
as sodium nitrate. However, in the inoculated cultures the growth was 
about 25 per cent better than in, the sterile, and much better than the in- 
oculated check and nitrate cultures. The plants in this culture were 
among the best of all the cultures. A part of these plants supphed v^ith 
hemoglobin were grown in very poor light and this may have had some 
detrimental influence, but even under such conditions they did exception- 
ally well. 

Casein 

Casein, like hemoglobin, is an anim.al protein, and might be thought to 
be unavailable for plant nutrition. Kelly (20) found that it may be 
readily ammonified by soil bacteria. The author's experiments show that 
in the sterile cultures it is favorable, about equal to sodium nitrate, and 
that in the inoculated cultures it is considerably better than the nitrate as 
a source of nitrogen. The inoculated cultures made a greater develop- 
ment than the sterile. 



B RICH AM— ASSIMILATION OF ORGANIC NITROGEN 189 

Linseed Meal 
That such products as Hnseed meal and cottonseed meal might be used 
as a source of nitrogen was suggested by Kelly (19). The results here 
reported show that plants furnished with linseed meal make a slightly bet- 
ter growth in the sterile cultures than the check plants, but not equal to 
that of the plants having sodium nitrate. The inoculated cultures with 
linseed meal were decidedly better than the sterile and also better than 
the inoculated nitrate cultures. 

Cottonseed Meal 
The results w4th cottonseed meal were very similar to those with lin- 
seed meal. That is, in the sterile cultures the growth was only slightly 
better than the check but in the inoculated it is markedly better, and the 
plants here were among the best of all the cultures. 

Malt 
The plants grown in the solution to which malt had been added m.ade 
approximately the same growth as those in the check in both the sterile 
and the inoculated cultures. This substance furnished practically no 
nitrogen, nor did the bacteria have any influence on the availability of 
the inoculated nitrate cultures. 

Creatin 
Creatin was used only in Series VII. This compound was of some 
value as a source of nitrogen, as indicated by the growth, which was 
somewhat better both in the sterile and in the inoculated cultures than the 
respective check cultures. There was little difference between them in 
growth in the sterile and in the inoculated cultures when the creatin was 
used as the source of nitrogen. 

Chemical Groups 
The inorganic nutrient salts, sodium nitrate and ammonium sulfate 
were both highly beneficial to plant growth as has already been stated, 
but they were excelled by some of the organic compounds. Among the 
organic compounds used were three purin derivatives. One, uric acid, 
was found available and decidedly beneficial, another guanin, was also 
found favorable to dent corn, while the third, caffein, containing three 
methyl groups, was quite toxic. The amids of the simple organic com- 
pounds are shown to contain nitrogen available for plant growth. Gly- 
cocoll and alanin are amids of acetic and propionic acids, respectively. 
Asparagin is a moiiamid of amido succinic acid and was one of the most 
favorable substances experimented with. Urea might be considered a 
diamido-carbonic acid, the simplest of all the organic acids. The albu- 
minoid substances peptone, casein and hemoglobin were also available 
for plant nutrition. 



190 SOIL SCIENCE 

The guanidin derivitives, guEinidin carbonate, guanidin nitrate and 
creatin appeared to furnish the plant with no nitrogen. The first two 
were noticeably toxic in their action, while creatin seemed free from toxic 
properties. 

Two compounds of the benzin ring group were used, benzamid and 
diphenylamin, the former with one, the latter with two benzin rings in 
the molecule. Both of these compounds were highly toxic to the Mays 
plants. The results with these two compounds are in accord with the 
work of Lutz (30) who has reported that benzylamin, diphenylamin, 
analin, and naphthylamin, members of the benzin series, were all toxic 
to the plants he employed. 

Of the ground seeds, cottonseed meal and linseed meal contained 
available nitrogen for the plants, while malt was of no value as a source 
of nitrogen. 

The results of this work and that of other investigators lead us to be- 
lieve that some substances containing organic nitrogen may be used as a 
source of this element for plants in general. The fact that plants under 
experiment can absorb some of the substances, without first being broken 
down, indicates that this can take place with the plants in the fields since 
they grow in soils containing manure or other decaying vegetable and ani- 
mal matter. Some of the substances then, in fertilizers, are directly as- 
similable by the plants and do not need to be ammonified and nitrified as 
is usually thought. Also, products probably occur in the intermediate 
stages of decomposition that may be directly utilized by plants. This is 
contrary to the general belief in agricultural practice that plants must be 
furnished with either ammonium compounds or nitrates. Nevertheless, 
most of the substances tried were utilized better or more rapidly when 
acted upon by B. subtilis. This is intelligible if B. subtilis causes ammoni- 
fication of such substances, since ammonium sulfate was better than 
sodium nitrate. 

Conclusions 

The results of the investigations reported in this thesis warrant the 
following conclusions : 

1. Zea Mays directly assimilates and uses the following organic nitro- 
genous compounds named in the order of their availability, asparagin, 
casein, cottonseed meal, hemoglobin, linseed meal, uric acid, peptone, 
guanin, alanin, urea, creatin, malt and glycocoll. 

2. The following organic nitrogenous compounds are toxic to the 
growth of Zea Mays: guanidin carbonate, guanidin nitrate, diphenylamin, 
caffein, and benzamid. Guanin is toxic to pop corn but not to dent corn. 

3. Eight organic substances which were directly available produced 
better growth when acted upon by B. subtilis, probably because of am- 
monification. These were peptone, guanin, alanin, linseed meal, cotton- 



BRIGHAM— ASSIMILATION OF ORGANIC NITROGEN 191 

seed meal, casein, hemoglobin and urea. The last showed this effect only 
with pop corn. 

4. The availability of the following substances was not increased by 
the action of B. subtilis: urea in the case of the dent corn, sodium nitrate, 
asparagin, ammonivim sulfate, uric acid, malt, creatin, glycocoll, and those 
compounds which were toxic. 

5. In the case of dent corn 6 substances were better than sodium 
nitrate ; cottonseed meal, linseed meal, casein, hamoglobin, uric acid, and 
asparagin. The following, though available, were not better than sodium 
nitrate: urea, peptone, guanin, alanin, and creatin. 

6. The different varieties of the same species of corn react differently 
with some nutrient substances. Guanin was toxic to pop corn but avail- 
able to dent corn. Peptone was better utilized by dent corn than by pop 
corn. 

7. The compounds of the benzin ring were found exceedingly toxic 
to the plants tried. 

8. Ammonium sulfate is a far better source of nitrogen for dent corn 
than sodium nitrate, and is surpassed only by casein and asparagin, when 
tested by the dry v/eight, and only by asparagin when tested by length of 
leaves produced. 

9. Generally, those organic compounds of high complexity in com- 
position are better after ammonification, while those of a low degree of 
complexity are not improved by ammonification. 

10. Very likely nitrification following ammonification would be detri- 
mental, since sodium nitrate was not equal to ammonium sulfate for dent 
corn. 

11. The method of measuring growth by length of leaves gave re- 
sults very nearly parallel to those obtained by determining the dry weight, 
and is much simpler. 

These conclusions apply to the two varieties of com plants used. 
Only experiments on other species and varieties will show how they react 
to these substances. 

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PLATE I 

Fig. 1. — One of the 1000-c.c. culture flasks used in growing the com plants ; show- 
the rolled cotton plug and through it passing the glass tube, in which the plant 
grew through the cotton plug. 

Fig. 2. — The 168 dent corn plants of Series VII. The flasks to the left of each 
niunber were sterile and to the right inoculated with B. suhtilis. The various 
compounds used are : O, distilled water; I, check; II, sodium nitrate; III, 
urea; IV, peptone; V, guanin; IX, alanin; X, ammonium sulfate; XI, aspara- 
gin; XIII, uric acid; XVI, hemoglobin; XVII, casein; XVIII, linseed meal; 
XIX, cottonseed meal; XX, malt; XXI, creatin. 



Brigham — Assimilation of Organic Nitrogen 



Plate I 



W^^- 


' 


! 


1 


■ 




i 


._-r' 


P 


m 


1 


CBM^^hH 


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f 
i 




1 


1 



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Soil Science 



Vol. ITT, No. 2 



Brigham — Assimilation of Organic Nitrogen 



Plate II 




Fig. 1 




Soil Science 



Fig. 2 



Vol. III. No. 2 



PLATE II 

Fig. 1. — Dent corn plants grown under sterile conditions of Series V, with differ-? 
ent forms of nitrogen added to the stock solution, namely, I, check ; II, sodium 
nitrate; III, urea; IV, peptone; V, guanin: VI, guanidin carbonate. 

Fig. 2. — Continuation of figure 1 : IX, alanin ; X, ammonium sulfate ; XI, asparagin ; 
XII, glycocoU; XIII, uric acid. 



LIBRARY OF CONGRESS 



002 756 470 7 



